CN108559008B - Quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced emission characteristic and preparation method thereof - Google Patents
Quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced emission characteristic and preparation method thereof Download PDFInfo
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 77
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 229920000669 heparin Polymers 0.000 title claims abstract description 63
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 35
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- 239000000243 solution Substances 0.000 claims description 44
- 239000000843 powder Substances 0.000 claims description 32
- 239000008367 deionised water Substances 0.000 claims description 28
- 229910021641 deionized water Inorganic materials 0.000 claims description 28
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
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- JLZUZNKTTIRERF-UHFFFAOYSA-N tetraphenylethylene Chemical compound C1=CC=CC=C1C(C=1C=CC=CC=1)=C(C=1C=CC=CC=1)C1=CC=CC=C1 JLZUZNKTTIRERF-UHFFFAOYSA-N 0.000 claims description 16
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- 238000003381 deacetylation reaction Methods 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 7
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- 229940127219 anticoagulant drug Drugs 0.000 claims description 3
- ZBKFYXZXZJPWNQ-UHFFFAOYSA-N isothiocyanate group Chemical group [N-]=C=S ZBKFYXZXZJPWNQ-UHFFFAOYSA-N 0.000 claims description 2
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- PUVAFTRIIUSGLK-UHFFFAOYSA-M trimethyl(oxiran-2-ylmethyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CC1CO1 PUVAFTRIIUSGLK-UHFFFAOYSA-M 0.000 claims description 2
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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Abstract
The invention discloses a quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced emission characteristics and a preparation method thereof. The method comprises the steps of firstly utilizing GTA to modify chitosan to prepare TMC with good water solubility and positive charge; grafting TPE molecules onto a quaternized chitosan molecular chain to obtain TPE-TMC; then the TPE-TMC and the natural anionic polymer heparin are subjected to electrostatic self-assembly in aqueous solution to prepare the composite nano fluorescent probe. The invention compounds heparin and TPE-TMC by an electrostatic self-assembly method, and the particle overcomes the inherent defects of the quaternized chitosan fluorescent probe after heparin modification, thereby obviously reducing the cytotoxicity, improving the blood compatibility and prolonging the in vivo circulation time. The fluorescent probe prepared by the invention has excellent biocompatibility and blood compatibility, has aggregation-induced emission characteristics, and has wide application prospect in the field of biological detection.
Description
Technical Field
The invention belongs to the field of nano fluorescent probes, and particularly relates to a quaternized chitosan/heparin nano fluorescent probe with aggregation-induced emission characteristics and a preparation method thereof.
Background
In recent years, cancer cases have increased year by year, and more people die, and in the course of cancer treatment, the diagnosis and detection of early cancer cells play a crucial role. Among a plurality of detection technologies, fluorescence detection is made by the characteristics of rapid reaction, high sensitivity, high resolution and the like, however, the traditional fluorescent molecules often have aggregation-induced quenching effect, and the application of the traditional fluorescent molecules is limited. In 2001, Tangbenzhou et al firstly discovered a class of molecules with aggregation-induced emission characteristics, and the molecules emit strong fluorescence in a solid state or an aggregation state, have the advantages of better photobleaching resistance, large Stokes shift and the like, and greatly promote the development of fluorescence detection technology.
Chitosan is a biodegradable natural polysaccharide and has excellent biocompatibility, anti-tumor activity, and the like, so the development of chitosan-based fluorescent probes has attracted attention in recent years. However, the chitosan-based fluorescent probe has poor water solubility, can only be dissolved in an acidic solution, and has certain limitation in practical application. The water solubility of the chitosan can be greatly enhanced by performing quaternization modification on the chitosan, and the like, but the problem of increased cytotoxicity caused by excessively high electropositivity can be caused.
Heparin is a sulfate anticoagulant drug with negative electricity, has the functions of anticoagulation, anti-inflammation, complement regulation, tumor growth inhibition and the like, and can be subjected to electrostatic compounding with quaternized chitosan with aggregation-induced emission characteristics, so that nanoparticles are formed through self-assembly. The electrostatic self-assembly method is simple to operate and quick in reaction, and the heparin-modified nano particles can reduce the electropositivity, greatly weaken the cytotoxicity, reduce the complement activation and prolong the circulation time in vivo. Therefore, the quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced emission characteristics, high biocompatibility, low cytotoxicity and in-vivo long retention is a new material in the field of biological detection.
Disclosure of Invention
The invention aims to provide a quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced emission characteristics and a preparation method thereof, so as to obtain the quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced emission, high biocompatibility, low cytotoxicity and long in-vivo retention.
The quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced emission characteristic is prepared by the following method: firstly, 2, 3-epoxypropyl trimethyl ammonium chloride (GTA) is used for modifying chitosan to prepare quaternized chitosan (TMC) with good water solubility and positive charge; grafting tetraphenyl ethylene (TPE) molecules with isothiocyanate groups onto a quaternized chitosan molecular chain to prepare a quaternized chitosan derivative (TPE-TMC) with aggregation-induced emission characteristics; then, the TPE-TMC and anticoagulant natural anionic polymer heparin are subjected to electrostatic self-assembly in an aqueous solution to form the quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced luminescence characteristics.
Specifically, the method comprises the following steps:
1) firstly, weighing chitosan powder, dissolving the chitosan powder in dilute acetic acid solution with the volume concentration of 2%, then adding GTA solution, reacting for 6 hours at 65 ℃, then dialyzing the reaction product to be neutral by using deionized water, and performing vacuum freeze-drying to obtain quaternized chitosan powder;
2) weighing the quaternized chitosan prepared in the step 1), swelling in a DMSO solvent, adding TPE, and reacting at 65 ℃ for at least 48 hours; dialyzing the mixture to be neutral by using deionized water, and freeze-drying to obtain TPE-TMC powder;
3) dissolving heparin powder in deionized water to obtain a heparin solution;
4) dissolving the TPE-TMC obtained in the step 2) in deionized water to prepare a TPE-TMC solution, dripping the heparin solution obtained in the step 3) into the TPE-TMC solution under stirring, and stirring to ensure that the heparin and the quaternized chitosan are subjected to electrostatic self-assembly in the water solution to form the quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced luminescence property.
In the technical scheme, the deacetylation degree of the chitosan is 80-95%, and the molecular weight is 20000-1000000.
The quaternization degree of the quaternized chitosan prepared in the step 1) is 15-50%.
The TPE grafting rate of the TPE-TMC prepared in the step 2) is 2-20%.
The invention has the beneficial effects that:
the invention takes amino in chitosan as a reaction site, modifies quaternary ammonium salt groups and aggregation-induced emission fluorescent groups on chitosan molecular chains, and then blends aqueous solution of the quaternary ammonium salt groups and aqueous solution of heparin to enable the quaternary ammonium salt groups and the aqueous solution of the heparin to be subjected to electrostatic self-assembly to form nano particles. The electrostatic composite modification of heparin obviously weakens the cytotoxicity of the nano particles, prolongs the circulation time of the nano particles in vivo and can prepare the nano fluorescent probe adapting to the microenvironment in vivo. The nano fluorescent probe prepared by the invention has high biocompatibility, low cytotoxicity and long retention in vivo, has aggregation-induced emission characteristics, photobleaching resistance and large Stokes shift, and can be applied to detection, tracing imaging and the like of cancer cells.
Drawings
FIG. 1 shows the structure of TPE-TMC and1HNMR spectrogram;
fig. 2 is a TEM image of quaternized chitosan/heparin composite nanoparticles.
Detailed Description
The invention is further illustrated below with reference to examples.
Example 1:
1) first, 1g of chitosan (molecular weight 20000, degree of deacetylation 80%) powder was dissolved in 2% by volume dilute acetic acid solution, and 0.25mL of GTA solution was added thereto, followed by reaction at 65 ℃ for 6 hours. Then, using deionized water to dialyze the reaction product to be neutral, carrying out vacuum freeze-drying to obtain quaternized chitosan powder, and analyzing the quaternization degree of the quaternized chitosan powder to be 15% by using a nuclear magnetic resonance spectrum;
2) 50mg of the quaternized chitosan prepared in step 1) were weighed out and swollen in 1ml of a DMSO solvent, then 5.0mg of TPE were added and reacted at 65 ℃ for 48 hours. Dialyzing the mixture to be neutral by using deionized water, and freeze-drying the mixture to obtain TPE-TMC powder, wherein the TPE grafting rate of the TPE-TMC powder is 5% by nuclear magnetic resonance spectrum analysis;
3) dissolving heparin powder with a certain mass into deionized water to obtain a heparin solution with the concentration of 2 mg/mL;
4) dissolving the TPE-TMC obtained in the step 2) in deionized water, preparing a TPE-TMC solution with the concentration of 4mg/mL, dripping an equal volume of the heparin solution obtained in the step 3) into the TPE-TMC solution under stirring, and stirring for 30 minutes to ensure that the heparin with negative charges and the quaternized chitosan with positive charges are subjected to electrostatic self-assembly in an aqueous solution to form a quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced luminescence characteristics, wherein the particle has charge controllability, low cytotoxicity, excellent blood compatibility and longer in-vivo circulation time;
5) the Average particle diameter (Z-Average) of the nano fluorescent probe obtained in the step 4) is 194.3 +/-0.7 nm and the Zeta potential is 17.6 +/-0.6 mV according to a dynamic light scattering method, but under the same condition, the Zeta potential of the TMC aqueous solution without heparin complexing is 36.1 +/-0.2 mV.
Because the surface of the cancer cell has negative charges, the TMC with high electropositivity is easy to interact with the cancer cell membrane and break the cancer cell membrane, and shows higher cytotoxicity. The electropositivity of the heparin composite modified nano particle is obviously reduced, the inherent defects of TMC are overcome, and the low cytotoxicity is realized. In addition, heparin has excellent anticoagulant property and the characteristic of reducing complement activation, and can greatly prolong the circulation time of the nano particles in vivo.
Example 2:
1) first, 1g of chitosan (molecular weight 30000, degree of deacetylation 95%) powder was dissolved in 2% dilute acetic acid solution, and 2.5mL of GTA solution was added and reacted at 65 ℃ for 6 hours. Then, using deionized water to dialyze the reaction product to be neutral, and carrying out vacuum freeze-drying to obtain quaternized chitosan powder with the quaternization degree of 50%;
2) 50mg of the quaternized chitosan prepared in step 1) were weighed out and swollen in 1ml of DMSO solvent, then 2.0mg of TPE were added and reacted at 65 ℃ for 48 hours. Dialyzing the mixture to be neutral by using deionized water, and freeze-drying the mixture to obtain TPE-TMC powder, wherein the grafting rate of the TPE is 2%;
3) dissolving heparin powder with a certain mass into deionized water to obtain a heparin solution with the concentration of 1 mg/mL;
4) dissolving the TPE-TMC obtained in the step 2) in deionized water, preparing a TPE-TMC solution with the concentration of 2mg/mL, dripping an equal volume of the heparin solution obtained in the step 3) into the TPE-TMC solution under stirring, and stirring for 30 minutes to ensure that the heparin with negative charges and the quaternized chitosan with positive charges are subjected to electrostatic self-assembly in an aqueous solution to form a quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced luminescence characteristics, wherein the particle has charge controllability, low cytotoxicity, excellent blood compatibility and longer in-vivo circulation time;
5) the Average particle diameter (Z-Average) of the nano fluorescent probe obtained in the step 4) is 146.4 +/-1.2 nm and the Zeta potential is 19.2 +/-0.6 mV according to the measurement of a dynamic light scattering method.
Example 3:
1) first, 1g of chitosan (molecular weight 34000, degree of deacetylation 95%) powder was dissolved in 2% dilute acetic acid solution, and 0.4mL of GTA solution was added and reacted at 65 ℃ for 6 hours. Then, using deionized water to dialyze the reaction product to be neutral, and carrying out vacuum freeze-drying to obtain quaternized chitosan powder with the quaternization degree of 20%;
2) 50mg of the quaternized chitosan prepared in step 1) were weighed out and swollen in 1ml of a DMSO solvent, then 50mg of TPE were added and reacted at 65 ℃ for 48 hours. Dialyzing the mixture to be neutral by using deionized water, and freeze-drying the mixture to obtain TPE-TMC powder, wherein the grafting rate of the TPE is 20%;
3) dissolving heparin powder with a certain mass into deionized water to obtain a heparin solution with the concentration of 0.2 mg/mL;
4) dissolving the TPE-TMC obtained in the step 2) in deionized water, preparing a TPE-TMC solution with the concentration of 0.4mg/mL, dripping an equal volume of the heparin solution obtained in the step 3) into the TPE-TMC solution under stirring, stirring for 30 minutes, and carrying out electrostatic self-assembly on the heparin with negative charges and the quaternized chitosan with positive charges in an aqueous solution to form a quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced luminescence characteristics, wherein the particle has charge controllability, low cytotoxicity, excellent blood compatibility and long in-vivo circulation time;
5) the Average particle diameter (Z-Average) of the nano fluorescent probe obtained in the step 4) is 148.4 +/-9.2 nm and the Zeta potential is 20.2 +/-0.5 mV according to the measurement of a dynamic light scattering method.
Example 4:
1) first, 1g of chitosan (molecular weight 1000000, degree of deacetylation 95%) powder was dissolved in 2% dilute acetic acid solution, and 0.5mL of GTA solution was added and reacted at 65 ℃ for 6 hours. Then, using deionized water to dialyze the reaction product to be neutral, and carrying out vacuum freeze-drying to obtain quaternized chitosan powder with the quaternization degree of 25%;
2) 50mg of the quaternized chitosan prepared in step 1) were weighed out and swollen in 1ml of DMSO solvent, then 3.5mg of TPE were added and reacted at 65 ℃ for 48 hours. Dialyzing the mixture to be neutral by using deionized water, and freeze-drying the mixture to obtain TPE-TMC powder, wherein the grafting rate of the TPE is 2.8%;
3) dissolving heparin powder with a certain mass into deionized water to obtain a heparin solution with the concentration of 2 mg/mL;
4) dissolving the TPE-TMC obtained in the step 2) in deionized water to prepare a TPE-TMC solution with the concentration of 3mg/mL, dripping an equal volume of the heparin solution obtained in the step 3) into the TPE-TMC solution under stirring, and stirring for 30 minutes to ensure that the heparin with negative charges and the quaternized chitosan with positive charges are subjected to electrostatic self-assembly in an aqueous solution to form a quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced luminescence characteristics, wherein the particle has charge controllability, low cytotoxicity, excellent blood compatibility and longer in-vivo circulation time;
5) the Average particle diameter (Z-Average) of the nano fluorescent probe obtained in the step 4) is 196.8 +/-1.3 nm and the Zeta potential is-31.7 +/-0.6 mV according to the measurement of a dynamic light scattering method.
Example 5:
1) first, 1g of chitosan (molecular weight 34000, degree of deacetylation 85%) powder was dissolved in 2% dilute acetic acid solution, and 0.4mL of GTA solution was added and reacted at 65 ℃ for 12 hours. Then, using deionized water to dialyze the reaction product to be neutral, and carrying out vacuum freeze-drying to obtain quaternized chitosan powder with the quaternization degree of 20%;
2) 50mg of the quaternized chitosan prepared in step 1) were weighed out and swollen in 1ml of DMSO solvent, then 3.5mg of TPE were added and reacted at 65 ℃ for 48 hours. Dialyzing the mixture to be neutral by using deionized water, and freeze-drying the mixture to obtain TPE-TMC powder, wherein the grafting rate of the TPE is 3%;
3) dissolving heparin powder with a certain mass into deionized water to obtain a heparin solution with the concentration of 0.2 mg/mL;
4) dissolving the TPE-TMC obtained in the step 2) in deionized water, preparing a TPE-TMC solution with the concentration of 0.2mg/mL, dripping an equal volume of the heparin solution obtained in the step 3) into the TPE-TMC solution under stirring, stirring for 30 minutes, and carrying out electrostatic self-assembly on the heparin with negative charges and the quaternized chitosan with positive charges in an aqueous solution to form a quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced luminescence characteristics, wherein the particle has charge controllability, low cytotoxicity, excellent blood compatibility and long in-vivo circulation time;
5) the Average particle diameter (Z-Average) of the nano fluorescent probe obtained in the step 4) is 85.75 +/-0.7 nm and the Zeta potential is-32.8 +/-1.1 mV, which are measured by a dynamic light scattering method.
Claims (5)
1. A quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced emission characteristics is characterized in that: the composite nano fluorescent probe is prepared by the following method: firstly, 2, 3-epoxypropyl trimethyl ammonium chloride (GTA) is used for modifying chitosan to prepare quaternized chitosan (TMC) with good water solubility and positive charge; grafting tetraphenyl ethylene (TPE) molecules with isothiocyanate groups onto a quaternized chitosan molecular chain to prepare a quaternized chitosan derivative (TPE-TMC) with aggregation-induced emission characteristics; then, the TPE-TMC and anticoagulant natural anionic polymer heparin are subjected to electrostatic self-assembly in an aqueous solution to form the quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced luminescence characteristics.
2. The method for preparing the quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced emission characteristics according to claim 1, comprising the steps of:
1) firstly, weighing chitosan powder, dissolving the chitosan powder in dilute acetic acid solution with the volume concentration of 2%, then adding GTA solution, reacting for 6 hours at 65 ℃, then dialyzing the reaction product to be neutral by using deionized water, and performing vacuum freeze-drying to obtain quaternized chitosan powder;
2) weighing the quaternized chitosan prepared in the step 1), swelling in a DMSO solvent, adding TPE, and reacting at 65 ℃ for at least 48 hours; dialyzing the mixture to be neutral by using deionized water, and freeze-drying to obtain TPE-TMC powder;
3) dissolving heparin powder in deionized water to obtain a heparin solution;
4) dissolving the TPE-TMC obtained in the step 2) in deionized water to prepare a TPE-TMC solution, dripping the heparin solution obtained in the step 3) into the TPE-TMC solution under stirring, and stirring to ensure that the heparin and the quaternized chitosan are subjected to electrostatic self-assembly in the water solution to form the quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced luminescence property.
3. The method for preparing a quaternized chitosan/heparin composite nano-fluorescent probe with aggregation-induced emission characteristics as claimed in claim 2, wherein the chitosan has a deacetylation degree of 80-95% and a molecular weight of 20000-1000000.
4. The method for preparing a quaternized chitosan/heparin composite nano fluorescent probe with aggregation-induced emission characteristics as claimed in claim 2, wherein the quaternization degree of the quaternized chitosan prepared in step 1) should be 15% -50%.
5. The method for preparing a quaternized chitosan/heparin composite nano-fluorescence probe with aggregation-induced emission characteristics as claimed in claim 2, wherein the TPE grafting ratio of the TPE-TMC prepared in step 2) is 2% -20%.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104845607A (en) * | 2015-03-20 | 2015-08-19 | 浙江大学 | Aggregation induced luminescence nanofluorescent probe and preparation method therefor |
| EP3060564A1 (en) * | 2013-10-24 | 2016-08-31 | Københavns Universitet | Azatriangulenium salts as pet-quenched fluorescent probes |
| CN106589163A (en) * | 2016-11-08 | 2017-04-26 | 浙江大学 | Quaternized chitosan fluorescent probe with aggregation-induced emission property and preparation method thereof |
| CN106632305A (en) * | 2016-12-09 | 2017-05-10 | 中南大学 | Fluorescent probe with water solubility and aggregation-induced emission effects, nanoparticle, preparation method and application |
| CN106893579A (en) * | 2017-01-19 | 2017-06-27 | 华南理工大学 | A kind of fluorescence probe and preparation method and purposes with aggregation-induced emission property |
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| CN106243244B (en) * | 2016-08-04 | 2019-05-14 | 浙江大学 | A kind of carboxyl chitosan namo fluorescence probe and preparation method thereof with aggregation-induced emission characteristic |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3060564A1 (en) * | 2013-10-24 | 2016-08-31 | Københavns Universitet | Azatriangulenium salts as pet-quenched fluorescent probes |
| CN104845607A (en) * | 2015-03-20 | 2015-08-19 | 浙江大学 | Aggregation induced luminescence nanofluorescent probe and preparation method therefor |
| CN106589163A (en) * | 2016-11-08 | 2017-04-26 | 浙江大学 | Quaternized chitosan fluorescent probe with aggregation-induced emission property and preparation method thereof |
| CN106632305A (en) * | 2016-12-09 | 2017-05-10 | 中南大学 | Fluorescent probe with water solubility and aggregation-induced emission effects, nanoparticle, preparation method and application |
| CN106893579A (en) * | 2017-01-19 | 2017-06-27 | 华南理工大学 | A kind of fluorescence probe and preparation method and purposes with aggregation-induced emission property |
Non-Patent Citations (2)
| Title |
|---|
| Long-Term Fluorescent Cellular Tracing by the Aggregates of AIE;Zhengke Wang 等;《J. Am. Chem. Soc》;20130513;第135卷(第22期);全文 * |
| 新型季铵盐壳聚糖纳米载药体系的研究;刘璠娜;《中国优秀博士论文全文数据库》;20080505;第17页、第37页 * |
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